Abstract We0078: Endothelial FAM222A Is Essential In Hypoxia-induced Revascularization Following Hind-Limb Ischemia

Background: Peripheral artery disease (PAD) and its associated tissue hypoxia induces compensatory responses, including angiogenesis and stimulation of endothelial cell (EC) cycling. Better understanding these compensatory responses will shed light on PAD and may help identify novel PAD therapeutic targets. We recently reported Family Sequence Similarity 222 Member A (FAM222A), a hypoxia-induced endothelial protein, controls angiogenesis in vitro and retinal revascularization in vivo . FAM222A regulation of angiogenesis remains incompletely understood and its role in hind limb ischemia (HLI) unexplored. Methods: FAM222A levels were analyzed in PAD patient transcriptomic datasets and human PAD gastrocnemius muscle biopsies. Mouse femoral artery ligation (FAL) models were used to study hypoxia-driven revascularization after siRNA or ASO FAM222A vs control knockdowns. ECs isolated from ischemic hindlimbs were used to assess transcriptional responses and RNA-seq to identify dysregulated pathways and upstream regulators. Angiogenesis, proliferation, reporter assays and cell cycle distribution (FACS analysis) were also performed. Mass spectrometry/precipitation enabled identification of FAM222A-interacting proteins. Results: FAM222A mRNA levels were induced significantly in ischemic gastrocnemius muscle of PAD patients and mice following FAL. Silencing endothelial FAM222A in vivo repressed revascularization after HLI, decreasing capillary formation, arteriogenesis and the number of proliferating ECs in the ischemic gastrocnemius, all vs controls. Expression of key endothelial cell cycling mediators was also decreased. Silencing FAM222A in vitro inhibited EC hypoxic responses, proliferation and caused G1 cell cycle arrest. RNA-Seq after siFAM222A treatment revealed significant repression of cell cycling and the DNA-damage response related pathways. FAM222A was found to bind to DNA replication factors, including mini-chromosome maintenance (MCM) complex and DNA damage response mediators. Loss of endothelial FAM222A resulted in replication stress, with decreased loading of the MCM replication machinery during G1 phase initiation and subsequent DNA damage response inhibition, with decreased H2A histone family member X(H2AX) activation. Conclusion: FAM222A is a novel determinant of compensatory hypoxia-driven revascularization responses in HLI, through its action regulating endothelial cell cycling and the DNA damage response.